Epidural administration of therapeutic compounds with sustained rate of release

ABSTRACT

A drug delivery system provides sustained-release delivery of therapeutic biologically active compounds administered epidurally. In the preferred embodiment the biologically active compound is an opioid, which is encapsulated within the non-concentric internal aqueous chambers or bilayers of multivesicular liposomes. The opioid is released over an extended period of time when the liposomes are introduced epidurally as a single dose for sustained analgesia.

THE FIELD OF THE INVENTION

[0001] This invention relates to controlled release of therapeuticcompounds from drug delivery systems. More particularly, this inventionrelates to epidural administration of therapeutic compounds withsustained rate of release from a liposome formulation. This inventionfurther relates to method of epidural catheter placement in a livingvertebrate.

BACKGROUND

[0002] Post-operative pain management is a serious issue for patientsand physicians, especially in the recovery room, as the patient iswaking up from the anesthesia. Too generous a dose of systemic opioidgiven in an attempt to control pain can potentially causelife-threatening respiratory depression. On the one hand, either toolittle or too late a dose of post-operative pain medication can resultin the patient waking up in intolerable severe pain. In addition, it hasbeen shown that poorly controlled post-operative pain followingabdominal or thoracic surgery inhibits ventilatory movement of the chestwall, abdomen, and diaphragm, (P. R. Bromage, Textbook of Pain, P. D.Wall, et al. (Eds.): Churchill Livingstone, 1989, pp 744-753) resultingin pulmonary atelectasis.

[0003] The existence of opioid receptors in the spinal cord wasdiscovered in the 1970's. Following initial clinical efficacy reports in1979 (M. Behar et al., Lancet 1:527-529, 1979), epidural opioidadministration has become very popular for post-operative pain control(T. I. Ionescu et al., Act. Anaesth. Belg. 40:65-77, 1989; C. Jayr etal., Anesthesiology 78:666-676, 1993; S. Lurie, et al. European Journalof Obstetrics and Gynecology and Reproductive Biology 49:147-153, 1993).Epidural opioids have the advantage of achieving good local analgesia atthe spinal level without the loss of locomotor or vasomotor control ordecreased level of consciousness.

[0004] Injectable opioids are widely used epidurally in post-operativeand post-partum settings. Post-operative and post-partum pain usuallylasts several days, but injectable opioids have relatively shortdurations of action (W. G. Brose et al., Pain 45:11-15, 1991; R. H.Drost et al., Arzneim-Forsch/Drug Res. 38:1632-1634, 1988; G. K. Gourlayet al., Pain 31:297-305, 1987). Thus, either continuous infusion orrepeated injections are required to maintain adequate pain control (J.W. Kwan, Am. J. Hosp. Pharm. 47 (Suppl 1):S18-23, 1990; J. S. Anulty,International Anesthesiology Clinics 28:17-24, 1990; R. S. Sinatra, TheYale Journal of Biology and Medicine 64:351-374, 1991. Continuousinfusion or repetitive injections further necessitate placement ofcatheter systems with or without attached infusion pumps, all of whichconsume expensive physician and nursing time for care and maintenance.Furthermore, repeated bolus injections or continuous infusions canresult in respiratory depression.

[0005] Late respiratory depression and apneic episodes are theside-effects of greatest concern in early studies (P. R. Bromage,Anesthesia and Analgesia 60:461-463, 1981; E. M. Camporesi, et al.,Anesthesia and Analgesia 62:633-640, 1983; T. L. Yaksh, Pain 11:293-346,1981). A recent prospective non-randomized study of epidural morphine in1085 patients who have undergone thoracic, abdominal, or orthopedicsurgeries estimated the rate of “respiratory depression” followingepidural morphine to be 0.9% (R. Stenseth et al., Acta Anaesthesiol.Scand. 29:148-156, 1985). As a comparison, the incidence of“life-threatening respiratory depression” in 860 patients given systemicmorphine (PO, IV, IM, SC) was 0.9% (R. R. Miller et al, Drug Effects inHospitalized Patients. John Wiley & Sons, New York, 1976). Prospective,randomized studies comparing epidural opioid versus systemic opioids (IMor IV) in high risk patients have shown that postoperative pain controlwith epidural opioid results in superior analgesia with decreasedincidence of post-operative complications (N. Rawal et al., Anesth.Analg. 63:583-592, 1984; M P. Yeager, et al. Anesth. 60: 729-736, 1987).

[0006] The sustained release of various therapeutic agents afterincorporation into liposomes, such as multivesicular liposomes, has beenwell documented both in vitro and in animals for intrathecal,subcutaneous, and intraperitoneal routes of administration, as well asin human patients for the intrathecal route of administration (S. Kim etal., J. Clin. Oncol. 11:2186-2193, 1993; V. Russack et al., Ann Neurol.34:108-112, 1993; and M. C. Chamberlain et al., Arch. Neurol.50:261-264, 1993). However, sustained release of epidurally administeredcompounds has heretofore been unknown in the art.

[0007] Therefore, the need exists for new and better methods foradministering opioids and other therapeutic compounds epidurally as asingle dose so as to achieve a sustained release rate at therapeuticallyeffective levels. The present invention addresses the limitations of theprior art by providing a sustained-release formulation of a therapeuticagent such as an opioid, that results in maximal analgesia immediatelyafter a single epidural dose and provides gradually decreasing analgesiaover the next several days.

DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 is a series of four graphs recording the analgesic effectin rats over time following a single epidural dose ofliposome-encapsulated morphine sulfate (DTC401) (open circles) or freemorphine sulfate (closed circles) for dosages (from top panel to bottompanel) of 10, 50, 175, or 250 μg. The intensity of analgesia isexpressed as “percent of maximum possible analgesia (%MPA)”. Each datapoint represents the average and standard error of mean (SEM) from 5 or6 animals.

[0009]FIG. 2 is a graph showing the peak-analgesia dose-response curvesas measured in rats after a single epidural dose of DTC401 (opencircles), free morphine sulfate (close circles), or after a singlesubcutaneous dose of free morphine sulfate (closed squares). The averagepeak %MPA±SEM was obtained from 5 or 6 animals.

[0010]FIG. 3 is a graph comparing the total-analgesic effect in rats [asmeasured by the area under the analgesia-time curves (AUC)] for singledoses of epidural DTC401 (open circles), or free morphine sulfate(closed circles). Each data point represents the average and standarderror of mean (SEM) from 5 or 6 animals.

[0011]FIG. 4 is a series of five graphs comparing the percent oxygensaturation of hemoglobin (SpO₂) in rats over time following singleepidural doses (from top panel to bottom panel) of 10, 50, 175, 1000, or2000 μg. of epidural DTC401 (open circles) or free morphine sulfate(closed circles). Each data point represents the average and standarderror of mean (SEM) from 5 animals except for 50 μg dose group wheren=3.

[0012]FIG. 5 is a graph showing the maximum respiratory-depressiondose-response curve in rats after a single epidural dose of DTC401 (opencircles) or free morphine sulfate (close circles). The lowest SpO₂achieved was plotted against epidural morphine dose. Each data pointrepresents the average and standard error of mean (SEM) from 5 animalsexcept for 50 μg dose group where n=3.

[0013]FIG. 6 shows two graphs comparing the pharmacokinetics in rats ofcerebrospinal fluid (top panel) and serum (bottom panel) following 250μg epidural administration of DTC401 (open circles) or free morphinesulfate (closed circles). Each data point represents average andstandard error of mean (SEM) from 3 or 4 animals.

SUMMARY OF THE INVENTION

[0014] Epidural administration of a therapeutic compound in a drugdelivery system provided surprisingly greater sustained release andduration of therapeutic effect compared to use of free therapeuticcompound.

[0015] Consequently, one aspect of the invention provides a method forthe sustained release of a therapeutic compound by utilizing a drugdelivery system administered epidurally to a vertebrate in need of suchtherapy.

[0016] Preferably, the vertebrate is a mammal such as a human. Invarious preferred embodiments, the drug delivery system is lipid based,especially when embodied as a multivesicular liposome.

[0017] The invention features the ability to allow sustained delivery ofvarious therapeutic compounds, which, in preferred embodiments,encompass opioids or opiate antagonists, to allow modulation ofanalgesia. Alternate embodiments allow delivery of such therapeuticcompounds as neurotrophic factors.

[0018] Furthermore, the use of a sustained-release formulation accordingto the method of the invention simplifies and reduces the over-all costof epidural analgesia by eliminating the need for continuous infusion,multiple bolus injections, or emplacement of catheters, and alsodecreases the likelihood of infection. Even in the presence of epiduralcatheters, reduced frequency of injection is advantageous.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention presents a lipid-based sustained-releasedrug delivery system for epidural delivery of a therapeutic compoundwith epidural efficacy, such as an opioid. By epidural administrationthe compounds are released to the central nervous system and thecerebrospinal spinal fluid without puncturing the dura and at asustained rate of release.

[0020] The term “sustained release” means that the therapeutic compound,when administered as a bolus dose encapsulated in the lipid-basedformulation is released over a longer period of time as compared toepidural administration of the same drug in free form as a bolusinjection. It does not necessarily mean that the concentration of thetherapeutic compound remains constant for a sustained period of time.Generally, following surgery or post-partum, the patient experiences adecreasing amount of pain as the days pass. The patient's need foranalgesia, therefore, also decreases over time. Using the method ofepidural drug delivery of this invention, a therapeutically effectivelevel of the therapeutic compound can be maintained in the cerebrospinalfluid and/or the serum over a period of several days, preferably fromabout 2 to about 7 days.

[0021] The term “therapeutic compound” as used herein means a chemicalcompound that has utility for modulating biological processes so as toachieve a desired effect in modulation or treatment of an undesiredexisting condition in a living being. The term therapeutic compoundembraces chemical non-proteinaceous drugs, such as antibiotic andanalgesics, as well as proteinaceous drugs, such as cytokines,interferons, growth factors, and the like.

[0022] Drug delivery systems are well known in the art. The presentinvention pertains to any sustained-release formulations such assynthetic or natural polymers in the form of macromolecular complexes,nanocapsules, microspheres, or beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, synthetic membranevesicles, and resealed erythrocytes. These systems are knowncollectively as dispersion systems. Dispersion systems are two-phasesystems in which one phase is distributed as particles or droplets in asecond phase. Typically, the particles comprising the system are about20 nm-50 μm in diameter. The size of the particles allows them to besuspended in a pharmaceutical solution and introduced to the epiduralspace using a needle or catheter and a syringe.

[0023] Materials used in the preparation of dispersion systems aretypically nontoxic and biodegradable. For example, collagen, albumin,ethyl cellulose, casein, gelatin, lecithin, phospholipids, and soybeanoil can be used in this manner. Polymeric dispersion systems can beprepared by a process similar to coacervation or microencapsulation. Ifdesired, the density of the dispersion system can be modified byaltering the specific gravity to make the dispersion hyperbaric orhypobaric. For example, the dispersion material can be made morehyperbaric by the addition of iohexol, iodixanol, metrizamide, sucrose,trehalose, glucose, or other biocompatible molecules with high specificgravity.

[0024] One type of dispersion system which can be used according to theinvention consists of a dispersion of the therapeutic agent in a polymermatrix. The therapeutic agent is released as the polymeric matrix, anddecomposes or biodegrades into soluble products that are excreted fromthe body. Several classes of synthetic polymers, including polyesters(Pitt, et al. In Controlled Release of Bioactive Materials, R. Baker,Ed., Academic Press, New York, 1980); polyamides (Sidman, et al.,Journal of Membrane Science, 7:227, 1979); polyurethanes (Master, etal., Journal of Polymer Science, Polymer Symposium, 66: 259, 1979);polyorthoesters (Heller, et al., Polymer Engineering Science, 21: 727,1981); and polyanhydrides (Leong, et al., Biomaterials, 7: 364, 1986)have been studied for this purpose. Considerable research has been doneon the polyesters of PLA and PLA/PGA. Undoubtedly, this is a consequenceof convenience and safety considerations. These polymers are readilyavailable, since they have been used as biodegradable sutures, and theydecompose into non-toxic lactic and glycolic acids (see, U.S. Pat. Nos.4,578,384; 4,785,973; incorporated by reference).

[0025] Solid polymeric dispersion systems can be synthesized using suchpolymerization methods as bulk polymerization, interfacialpolymerization, solution polymerization, and ring opening polymerization(Odian, G., Principles of Polymerization, 2nd ed., John Wiley & Sons,New York, 1981). Using any of these methods, a variety of differentsynthetic polymers having a broad range of mechanical, chemical, andbiodegradable properties are obtained; the differences in properties andcharacteristics are controlled by varying the parameters of reactiontemperatures, reactant concentrations, types of solvent, and reactiontime. If desired, the solid polymeric dispersion system can be producedinitially as a larger mass which is then ground, or otherwise processed,into particles small enough to maintain a dispersion in the appropriatephysiologic buffer (see, for example, U.S. Pat. Nos. 4,452,025;4,389,330; 4,696,258; incorporated by reference).

[0026] If desired, a therapeutic compound can be incorporated into anon-disperse structure which is epidurally implanted by surgical ormechanical means. A non-disperse structure is one having a definedoverall shape, such as a slab, cylinder or sphere. The mechanism ofrelease of therapeutic agent from biodegradable slabs, cylinders, andspheres has been described by Hopfenberg (in Controlled ReleasePolymeric Formulations, pp. 26-32, Paul, D. R. and Harris, F. W., Eds.,American Chemical Society, Washington, D.C., 1976). A simple expressiondescribing additive release from these devices where release iscontrolled primarily by matrix degradation is:

M _(t) /M _(∞)=1−[1−k _(o) t/C _(o)α]^(n)

[0027] where n=3 for a sphere, n=2 for a cylinder, and n=1 for a slab.The symbol α represents the radius of a sphere or cylinder or thehalf-thickness of a slab. M_(t) and M_(∞) are the masses of drugreleased at time t and at infinity, respectively.

[0028] Any of the known lipid-based drug delivery systems can be used inthe practice of the invention. For instance, multivesicular liposomes(MVL), multilamellar liposomes (also known as multilamellar vesicles or“MLV”), unilamellar liposomes, including small unilamellar liposomes(also known as unilamellar vesicles or “SUV”) and large unilamellarliposomes (also known as large unilamellar vesicles or “LUV”), can allbe used so long as a sustained release rate of the encapsulatedtherapeutic compound can be established. In the preferred embodiment,however, the lipid-based drug delivery system is a multivesicularliposome system. The method of making controlled release multivesicularliposome drug delivery systems is described in full in U.S. patentapplication Ser. No. 08/352,342 filed Dec. 7, 1994, and Ser. No.08/393,724 filed Feb. 23, 1995 and in PCT Application Serial Nos.US94/12957 and US94/04490, all of which are incorporated herein byreference in their entireties.

[0029] The composition of the synthetic membrane vesicle is usually acombination of phospholipids, usually in combination with steroids,especially cholesterol. Other phospholipids or other lipids may also beused.

[0030] Examples of lipids useful in synthetic membrane vesicleproduction include phosphatidylglycerols, phosphatidylcholines,phosphatidylserines, phosphatidylethanolamines, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine,distearoylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol.

[0031] In preparing vesicles containing a therapeutic agent, suchvariables as the efficiency of drug encapsulation, lability of the drug,homogeneity and size of the resulting population of vesicles,drug-to-lipid ratio, permeability, instability of the preparation, andpharmaceutical acceptability of the formulation should be considered.(Szoka, et al., Annual Revievs of Biophysics and Bioengineering, 9:467,1980; Deamer, et al., in Liposomes, Marcel Dekker, New York, 1983, 27;Hope, et al., Chem. Phys. Lipids, 40: 89, 1986).

[0032] The use of lipid-based formulations of opioids has beeninvestigated by others with limited success and none has beeninvestigated via epidural route. For instance, preparation and in vitroactivity of liposome encapsulated opioids has been studied (F. Reig, etal., J. Microencapsulation 6:277-283, 1989) without any epidural in vivoinvestigation. In addition, antinociception and side effects ofalfentanil encapsulated in a liposome formulation and introduced byspinal delivery into rats has been explored (M. S. Wallace et al.,Anesth Analg. 79:778-786, 1994; C. M. Bernards et al., Anesthesiology77:529-535, 1992). However, neither the pharmacokinetics nor thepharmacodynamics of these compounds were sufficiently different fromthose of the standard opioids to warrant their use in clinical practice.These studies did not explore sustained-release formulations of opioidsgiven via the epidural route.

[0033] The lipid-based drug delivery system incorporating thetherapeutic compound can be delivered as a single dose, for instance,via an epidural catheter. In the preferred embodiment, however, thelipid-based drug delivery system is injected as a single dose into theepidural space surrounding the spinal cord using a small gauge needle sothat emplacement of a catheter is avoided. Preferably, an 18 gauge to 25gauge needle is used.

[0034] A representative list of the therapeutic compounds useful forepidural delivery includes the opiates morphine, hydromorphone, codeine,hydrocodone, levorphanol, oxycodone, oxymorphone, diacetyl morphine,buprenorphine, nalbupine, butorphanol, pentazocine, methadone, fentanyl,sufentanyl and alfentanyl. In addition, opiate antagonists, such asnaloxone and naltrexone, can be administered epidurally using the methodof the invention to reverse or antagonize opiate effect.

[0035] Peptides and peptidomimetics that bind to one or moreneuroreceptors such as the delta opioid, mu-opioid, kappa opioid andepisilon-opioid receptors are considered opioids and can be administeredfor therapeutic effect according to the method of the invention. Suchcompounds include enkephalins, endorphins, casomorphin, kyotorphin, andtheir biologically active fragments. As used herein, the term“biologically active fragment” means any portion of a therapeuticcompound that substantially retains the biological activity of thecomplete therapeutic molecule. One skilled in the art will know, or caneasily determine, whether a fragment substantially retains thebiological activity of the whole molecule.

[0036] In addition to opioids, a number of compounds having therapeuticutility when administered epidurally at a sustained rate can also beused in the practice of the method of the invention. These compoundsinclude neurotrophic factors, such as insulin-like growth factor,ciliary neurotrophic factor and nerve growth factors; neurotransmittersand their antagonists, such as dopamine, epinephrine, norepinephrine,and gamma-amino butyric acid; local anesthetics, such as tetracaine,lidocaine, bupivacaine, and mepivacaine; substance P and relatedpeptides; and alpha-2-receptor agonists, such as clonidine anddexmedetomidine. Further, co-administration of local anesthetics such aslidocaine, bupiracaine, and tetracaine can increase efficacy of epiduralopioids.

[0037] In the present invention, it is shown that a lipid-based drugdelivery system incorporating an opioid, such as morphine sulfate, hasminimal potential for respiratory depression as measured by percentdecrease in hemoglobin oxygen saturation (SpO₂) from the maximum bloodoxygen saturation or baseline value prior to administration of the drug,compared to epidural administration of the free drug. One skilled in theart will appreciate that blood oxygen content can readily be measured bysuch commercially available devices as a pulse oximeter.

[0038] It is also shown that a single dose of sustained release opioidformulated in a multivesicular liposome composition and administeredepidurally results in prolonged duration of analgesia, with the peakcisternal CSF concentration of the therapeutic drug occurring within 60minutes after a single epidural dose and then gradually decreasing overthe next several days, for instance up to eight days. Although the peakCSF concentration was decreased compared with that following epiduraladministration of free morphine sulfate, the total analgesia delivered(as shown, for example, by the area under the curve (AUC) in FIGS. 1, 3,and Table 1) was increased many fold compared to epidurally deliveredfree morphine sulfate. For instance, in rats there were 17- and 3.1-foldreductions in the peak serum and CSF morphine concentrations,respectively, but CSF AUC was increased 2.8 fold following epiduraladministration of 250 μg of morphine sulfate encapsulated inmultivesicular liposomes (DTC401) compared to an identical dose ofunencapsulated morphine sulfate.

[0039] Because of the reduction in the peak serum and CSF concentrationsof morphine, there was no respiratory suppression with the controlledrelease of epidurally administered morphine; whereas epidurallyadministered free morphine did cause respiratory suppression at highdosages.

[0040] The chief advantages of the present invention are threefold.First, the method of epidural delivery of a single dose of sustainedrelease compound provides the advantage that the patient experiences areduced risk of dose-related adverse effects, such as respiratorydepression normally associated with bolus epidural injections orinfusions of a therapeutic compound. Second, by administration of thetherapeutic compound epidurally rather than directly into thecerebrospinal fluid, the therapeutic compound does not migrate all overthe brain and spinal cord, and a therapeutically effective dosage of thetherapeutic compound is released locally into the epidural space over anextended period of time, for instance up to eight days. And finally,prolonged analgesia is obtained without multiple injections orcontinuous infusions.

[0041] One skilled in the art will comprehend that the period of timeover which a therapeutic rate of release is maintained in the practiceof the invention will vary depending upon the disease state to betreated, the characteristics of the therapeutic compound and thesustained-release drug delivery system, and the total amount of thecompound encapsulated and administered to the patient.

[0042] The term “therapeutically effective” as it pertains to thecompositions of the invention means that the therapeutic compound isreleased from the drug delivery system at a concentration sufficient toachieve a particular medical effect for which the therapeutic agent isintended. For instance, if the therapeutic compound is an opioid, thedesirable medical effect is analgesia without respiratory depression.Exact dosages will vary depending upon such factors as the particulartherapeutic compound and desirable medical effect, as well as patientfactors such as age, sex, general condition, and the like. Those ofskill in the art can readily take these factors into account and usethem to establish effective therapeutic concentrations without resort toundue experimentation.

[0043] For instance, the dosage range appropriate for epiduraladministration of morphine sulfate to a human includes the range of 1 mgto 60 mg. More potent compounds can require dosages as low as 0.01 mgand less potent compounds can require 5000 mg. While doses outside theforegoing dose range may be given, this range encompasses the breadth ofuse for practically all the therapeutic substances contemplated foradministration by an epidural route.

[0044] Previously published methods of epidural placement in ratsinvolves drilling a hole through a lumbar vertebral bone and pushing acatheter 1 cm up the epidural space. The present invention enablesplacement of a catheter from above (i.e., from the cervical region)without the trauma of a surgical procedure. Also, the catheter tip canbe placed at any location along with the vertebral column rather thanbeing restricted to the lumbar region as described in the prior art.This method of catheter placement from above is also applicable toanimals other than rats, such as rabbits, dogs, and humans.

[0045] The following examples illustrate the manner in which theinvention can be practiced. It is understood, however, that the examplesare for the purpose of illustration and the invention is not to beregarded as limited to any of the specific materials or conditionstherein.

EXAMPLE 1

[0046] A. Preparation of Multivesicular Liposomes Encapsulating MorphineSulfate (DTC401) in the Presence of a Hydrochloride

[0047] Step 1) In a clean one-dram glass vial (1.3 cm inner diameter×4.5cm height), were placed 1 ml of a chloroform (Spectrum Corp., Gardena,Calif.) solution containing 9.3 μmoles of dioleoyl lecithin (AvantiPolar Lipids, Alabaster, Ala.), 2.1 μmoles of dipalmitoylphosphatidylglycerol (Avanti Polar Lipids), 15 μmoles of cholesterol(Avanti Polar Lipids), and 1.8 μmoles of triolein (Sigma). This solutionis referred to as the lipid component.

[0048] Step 2) One ml of an aqueous solution containing 20 mg/ml ofmorphine sulfate (Sigma Chemical Co., St. Louis, Mo.) and 0.1N ofhydrochloric acid, was added into the above one-dram glass vialcontaining the lipid component.

[0049] Step 3) For making the water-in-oil emulsion, the glass vialcontaining the mixture of “Step 2” was sealed and attached horizontallyto the head of a vortex shaker (Catalogue #S8223-1, American ScientificProducts, McGaw Park, Ill.) and shaken at maximal speed for 6 minutes.

[0050] Step 4) For making the chloroform spherules suspended in water,the water-in-oil emulsion obtained from “Step 3” was divided in equalvolume and expelled rapidly through a narrow-tip Pasteur pipette intoeach of two one-dram glass vials (1.3 cm inner diameter×4.5 cm height),each containing 2.5 ml water, glucose (32 mg/ml), and free-base lysine(40 mM) (Sigma). Each vial was then sealed, attached to the head of thesame vortex shaker as used in “Step 3” and shaken for 3 seconds atmaximal speed to form chloroform spherules.

[0051] Step 5) To obtain the multivesicular liposomes, chloroformspherule suspensions produced in the two vials of “Step 4” were pouredinto the bottom of a 250 ml Erlenmeyer flask containing 5 ml of water,glucose (32 mg/ml), and free base lysine (40 mM). With the flask kept at37° C. in a shaking water bath, a stream of nitrogen gas at 7 L/minutewas flushed through the flask to slowly evaporate chloroform over 10-15minutes. The liposomes were then isolated by centrifugation at 600×g for5 minutes; and washed three times in 0.9% NaCl solution.

[0052] B. Preparation of Formulations

[0053] Prior to epidural injection, preparations of the DTC401 and acontrol of unencapsulated (“free”) morphine sulfate were adjusted sothat 50 μl contained the dose of 10, 50, 175, 250 or 1000 μg. Inaddition, a preparation of MVLs containing a 2000 μg dose of morphinesulfate to be used in a study of respiratory suppression was formulatedin a 75 μl volume for injection. The concentration of morphine in thevarious liposome formulations was determined by dissolving 50 μl of eachpreparation with 1 ml of isopropyl alcohol, followed by dilution inwater. The morphine concentration was assayed with HPLC using apublished method (S. P. Joel et al., Journal of Chromatography430:394-399, 1988). For the placebo control, a blank multivesicularliposome composition was made by substituting glucose in place ofmorphine sulfate.

EXAMPLE 2

[0054] A. Animal Preparation

[0055] Six- to 8-week old male Sprague-Dawley rats weighing 205-254 g(Harlan Sprague-Dawley, San Diego, Calif.) were housed, 1 or 2 per cage,in a temperature-controlled environment with an alternating 12-hourlight and darkness cycle and given unrestricted access to food andwater. Prior to each study, animals were habituated to the environment.Each animal was studied only once. All animals were maintained inaccordance with guidelines of the Committee on Care and Use ofLaboratory Animals of the Institute of Laboratory Animal Resources,National Research Council.

[0056] B. Epidural Catheterization

[0057] Caudal epidural catheterization of rats was performed as follows:Halothane anesthesia was induced and the animals were placed instereotaxic recumbency 7 cm in height. The head was flexed, taking carethat animals maintained normal breathing. A short-beveled 19-gaugeneedle was inserted at an angle of approximately 170° to the spine justcaudad to the occipital crest in the midline with needle bevel facingdown. The needle was advanced caudad towards the C1 vertebra until theneedle tip touched the spinous process or posterior lamina of C1. Theneedle tip was walked carefully to the ventral edge of the posteriorlamina. At this point, a slight give was felt and the needle wasadvanced 1-2 mm further. Care was taken not to let the needle penetratethe dura. Accidental violation of the dura can be determined by a flashof cerebrospinal fluid (CSF) through the hub of the needle or throughthe subsequently-placed catheter.

[0058] A polyethylene catheter (PE-10; length: 12 cm, i.d.: 0.28 mm;volume: 7.4 μl (Becton Dickinson, Sparks, Md.) was threaded through theneedle into the dorsal epidural space. The catheter was advanced slowlythrough the needle and stopped at the approximate level of L1, 8 cm fromC1. The exposed portion of the catheter was subcutaneously tunneledunder the scalp and fixed with a purse-string 3-0 silk suture. Finally,the catheter was flushed with 10 μl of normal saline and plugged with astainless steel wire. The procedure from initiation of anesthesia tosutures lasted approximately 10 to 15 minutes. Animals were allowed torecover and were observed for a period of 60 minutes. Only those animalsthat completely recovered from the procedure were used in the followingstudies.

[0059] C. Antinociception

[0060] Baseline values of nociception following placement of theepidural catheter were determined by subjecting the animals to standardhot plate (52.5±0.5° C.) testing as described in M. S. Wallace et al.,(Anesth. Analg. 79:778-786, 1994). Response latency to nociception (inseconds) was measured from the time when the animals were placed on thehot plate to the time when they either licked their hind paw or jumped.The baseline (pretreatment) response latency value was defined as 0% ofthe maximum possible analgesia (MPA) in each experimental animal. Theneach animal was injected epidurally with 50 μl of either DTC401containing doses of epidural morphine ranging from 10 μg to 250 μg,unencapsulated morphine sulfate solution, or control MVL blanks. Theantinociceptive effect of subcutaneously administered morphine sulfatewas also determined in a dose range of 250 μg to 1 mg. Followingepidural administration of the test solutions via the catheter emplantedas described above, the epidural catheter was flushed with 10 μl of 0.9%sodium chloride.

[0061] The animals were then subjected to hot plate testing again formeasurement of antinociceptive effect at specific time points: 0.5, 1,2, 3, 4, 6, 12, and 24 hours following administration for unencapsulatedmorphine sulfate and 0.5, 1, 6 hours and 1, 2, 3, 4, 5, 6, 7, and 8 daysfollowing administration for both DTC401 and the MVL blanks.Antinociception was determined in 5 or 6 animals for each dose and eachdrug. To prevent tissue damage to the footpads, a cutoff time of 60seconds was used. Accordingly, 100% MPA was defined as antinociceptionlasting ≧60 seconds. A latency interval of 10±2 to 60 secondscorresponding to an MPA of 0% to 100%, respectively. was sensitive fordemonstrating dose-response in the studied dose range.

[0062] Efficacy and respiratory depression curves were plotted as afunction of time for each dose administered. Hot plate responses werecalculated as a percentage of the maximum possible analgesia (%MPA) asdescribed in Wallace et al. (supra):${\% \quad {MPA}} = {\frac{{{Postdrug}\quad {latency}} - {{Predrug}\quad {latency}}}{{{Cutoff}\quad {latency}} - {{Predrug}\quad {latency}}} \times 100\%}$

[0063] All areas under the curves were calculated by the trapezoidalrule to the last data point using the RSTRIP computer program[Micromath, Salt Lake City, Utah].

[0064] One-way analysis of variance (ANOVA) was used to separatelydetermine dose dependency for the different drug formulations androutes; whereas two-way ANOVA was used for comparison betweenformulations at an equal dose. The Newman-Keuls test was performed onall ANOVA analyses to determine statistical significance; p<0.05 wasconsidered statistically significant for all tests. All data isdisplayed as the mean±standard error of the mean (SEM).

[0065] As shown by the data in FIG. 1, the epidural administration ofDTC401 resulted in equivalent onset of analgesia, but the duration ofanalgesia was significantly prolonged compared to epidurallyadministered free morphine sulfate. Epidural injection of control MLVblanks showed no demonstrable antinociceptive effect (data not shown).The peak analgesic effects of epidural DTC401 and epidural andsubcutaneous morphine sulfate were dose dependent, as shown in FIG. 2,with the peak-analgesia potency of epidural free morphine sulfate beinggreater than that of epidural DTC401, which is substantially greaterthan that of subcutaneously administered free morphine sulfate (p<0.05for each comparison).

[0066] Substantial prolongation of analgesic effects in animals givenepidural DTC401 is seen readily in FIG. 1 as well as by the largearea-under-the-curve values (AUC) for DTC401 in FIG. 3. At the dose of250 μg, which produced peak effects close to 100% MPA for both DTC 401and free morphine sulfate, the time to decrease to 50% MPA was 3.4 daysfor DTC 401 compared to 0.17 day for morphine sulfate.

[0067] D. Respiratory Depression

[0068] Respiratory depression was quantified by pulse oximetry. Theanimals were removed from their cages, placed in polystyrene ratrestraints (Plas Labs, Lansing, Mich.) and allowed to acclimate for 5minutes. Oxygen saturation was determined at baseline and following asingle epidural bolus of morphine sulfate or DTC40 1 at specific timepoints by placing a pulse oximeter probe on the right hind paw (OhmetaMedical Systems, model 3740, Madison, Wis.). The doses of DTC401 andfree morphine sulfate ranged from 10 to 2000 μg. Pulse oximetry wasperformed on 5 to 6 animals at each data point, except for the 50 μgdose where 3 animals were used. The pulse oximetry values of percenthemoglobin oxygen saturation (SpO2) were monitored continuously in realtime. The maximum value obtaining during the 3-minute recording periodwas defined as oxygen saturation.

[0069]FIG. 4 depicts the time course of percent oxygen saturation ofhemoglobin (SpO₂) as measured by the pulse oximeter at various doses ofDTC401 and morphine sulfate. There was a dose-dependent increase inrespiratory depression with increasing doses of morphine sulfate asshown in FIG. 5; whereas minimal respiratory depression was produced bythe same doses of DTC401. On the other hand, the maximum decreases inSpO₂ were observed within 1 hour following epidural administration offree morphine sulfate or DTC401, and no delayed respiratory depressionwas seen with either formulation. The difference between morphinesulfate and DTC401 on peak respiratory depression was statisticallysignificant (p<0.01).

[0070] E. Pharmacokinetics

[0071] The pharmacokinetic studies were done by measuring morphineconcentrations in peripheral blood and in CSF at appropriate time pointsfollowing a single 250 μg epidural dose of DTC401 or free morphinesulfate. Samples were drawn at 0.5, 1 hours, and 1, 3, 5, 8 daysfollowing epidural administration as described above of DTC401 and at0.5, 1, 3, 6, 12, 24 hours following epidural administration of freemorphine sulfate. A set of 3 or 4 animals were anesthetized usinghalothane, and CSF and blood samples were collected via cisternal tapand cardiac puncture, respectively. The animals were then sacrificed byoverdose of halothane. Serum was separated from blood by centrifugationand stored along with CSF samples at −80° C. until further analysis byradioimmunoassay (RIA).

[0072] Morphine concentrations in serum and CSF were determined using acommercially available RIA kit highly specific for morphine[Coat-A-Count™ Serum Morphine, Diagnostic Products Corp., Los Angeles,Calif.] as suggested by the manufacturer. All measurements were done induplicate.

[0073]FIG. 6 shows the concentrations of cisternal CSF and serummorphine in animals injected with 250 μg of free morphine sulfate orDTC401. Table 1 summarizes the pharmacokinetic parameters. The peak CSFand serum morphine concentrations following epidural administration ofDTC401 were, respectively, 32% and 5.9% of that following morphinesulfate. The terminal CSF half-life (β) for DTC401 was 82 hours comparedto 2.6 hours for morphine sulfate. The CSF area under the curve (AUC)was increased 2.7 times for DTC401 compared to morphine sulfate, but theplasma AUC was very similar. Half-lives were calculated by fitting thepharmacokinetic curves to a biexponential function. The RSTRIP programwas used to perform the curve fitting by iterative nonlinear regression.

EXAMPLE 3

[0074] Larger Scale preparation of DTC401

[0075] Step 1) Into a clean stainless steel 50 ml centrifuge tube wereplaced 5 ml of a chloroform solution containing 46.5 μmoles of dioleoylphosphatidylcholine (Avanti Polar Lipids), 10.5 μmoles of dipalmitoylphosphatidylgylycerol (Avanti Polar Lipids), 75 μmoles of cholesterol(Sigma Chemical Co.), 9.0 μmoles of triolein (Avanti Polar Lipids). Thissolution is referred to as the lipid component.

[0076] Step 2) Five ml of an aqueous solution containing 20 mg/ml ofmorphine sulfate pentahydrate (Mallinckrodt Chemical Inc.) and 0.1 N ofhydrochloric acid was added into the above stainless steel centrifugetube containing the lipid component.

[0077] Step 3) For making the water-in-oil emulsion, the mixture of Step2 was stirred with a TK mixer (AutoHomoMixer, Model M, Tokushu Kika,Osaka, Japan) at a speed of 9000 revolution per minute (rpm) for 9minutes.

[0078] Step 4) For making the chloroform spherules suspended in water,25 ml of solution containing 4 percent glucose and 40 mM lysine in waterwas added to the water-in-oil emulsion of Step 3 and then mixed at aspeed of 3500 rpm for 120 seconds.

[0079] Step 5) To obtain the multivesicular liposomes, the chloroformspherule suspension in the centrifuge tube was poured into the bottom ofa 1000 ml Erlenmeyer flask containing 25 ml of 4 percent glucose and 40mM lysine in water. With the container kept at 37 C. in a shaking waterbath, a stream of nitrogen gas at 7 L/minute was flushed through theflask to slowly evaporate chloroform over 20 minutes. The liposomes werethen isolated by 4-fold dilution of the suspension with normal salineand centrifugation of the suspension at 600×g for 5 minutes; thesupernatant was decanted, and the liposome pellet was resuspended in 50ml of normal saline. The liposomes were isolated again by centrifugationat 600×g for 5 minutes. The supernatant was again decanted and thepellet was resuspended in normal saline.

[0080] The foregoing description of the invention is exemplary forpurposes of illustration and explanation. It should be understood thatvarious modifications can be made without departing from the spirit andscope of the invention. Accordingly, the following claims are intendedto be interpreted to embrace all such modifications. TABLE 1Pharmacokinetic parameters following 250-μg epidural injection DTC401 MSCmax (ng/ml), CSF 1960 ± 1280 6060 ± 3590 Cmax (ng/ml), Serum 86 ± 201460 ± 97  t½ α (hr), CSF 5.0 0.85 t½ β (hr), CSF 82 2.6 t½ α (days),Serum 0.48 0.68 t½ β (hr), Serum 49 5.0 AUC (ng*days*ml⁻¹) CSF 1170 432AUC (ng*days*ml⁻¹) Serum 53 58

[0081] MS, morphine sulfate; Cmax, maximum concentration; t½ α, initialhalf-life; t½ β, terminal half-life; AUC, area under the curve.

What is claimed is:
 1. A method for epidural administration to avertebrate of a therapeutic compound comprising encapsulating thecompound in a drug delivery system having a sustained release rate ofthe compound, and introducing the drug delivery system epidurally to thevertebrate.
 2. The method of claim 1 wherein the vertebrate is a mammal.3. The method of claim 2 wherein the mammal is a human.
 4. The method ofclaim 1 wherein the delivery system is administered as a single dose. 5.The method of claim 1 wherein the drug delivery system is a dispersionsystem.
 6. The method of claim 5 wherein the drug delivery systemcomprises a lipid-based formulation.
 7. The method of claim 5 whereinthe drug delivery system comprises a liposome formulation.
 8. The methodof claim 5 wherein the drug delivery system comprises a multivesicularliposome formulation.
 9. The method of claim 7 or 8 wherein thetherapeutic compound is an opioid.
 10. The method of claim 7 or 8wherein the therapeutic compound is a peptide or peptidomimetic.
 11. Themethod of claim 9 wherein the therapeutic compound is morphine sulfate.12. The method of claim 9 wherein the therapeutic compound ishydromorphone.
 13. The method of claim 7 or 8 wherein the therapeuticcompound is selected from the group consisting of codeine, hydrocodone,levorphanol, oxycodone, oxymorphone, diacetyl morphine, buprenorphine,nalbupine, butorphanol, pentazocine, methadone, fentanyl, sufentanyl,and alfentanyl.
 14. The method of claim 7 or 8 wherein the therapeuticcompound is selected from the group consisting of enkephalins,endorphins, casomorphin, kyotorphin, and their bioactive fragments. 15.The method of claim 7 or 8 wherein the therapeutic compound is an opiateantagonist.
 16. The method of claim 15 wherein the opiate antagonist isselected from the group consisting of naloxone and naltrexone.
 17. Themethod of claim 7 or 8 wherein the therapeutic compound is aneurotrophic factor.
 18. The method of claim 17 wherein the neurotrophicfactor is selected from the group consisting of insulin-like growthfactor, ciliary neurotrophic factor, nerve growth factors, dopamine,epinephrine, norepinephrine, gamma-amino butyric acid and neostigmine.19. The method of claim 1 wherein the drug delivery system is introducedvia an epidural catheter.
 20. The method of claim 19 wherein theepidural catheter is inserted downwards from the cervical region. 21.The method of claim 1 wherein the drug delivery system is introduced viaa hypodermic needle inserted into the epidural space.
 22. A method forameliorating respiratory depression in a patient administered ananalgesic compound comprising epidurally administering to the patient asingle dose of the analgesic compound encapsulated in a sustainedrelease liposome formulation.
 23. The method of claim 22 wherein theliposome formulation comprises multivesicular liposomes.
 24. The methodof claim 23 wherein the analgesic compound is an opioid.
 25. The methodof claim 24 wherein the opioid is morphine sulfate.
 26. The method ofclaim 24 wherein the opioid is selected from the group consisting ofhydromorphone, codeine, hydrocodone, levorphanol, oxycodone,oxymorphone, diacetyl morphine, buprenorphine, nalbupine, butorphanol,pentazocine, methadone, fentanyl, sufentanyl and alfentanyl.
 27. Themethod of claim 25 wherein the dose contains from about 1 mg to 60 mg ofmorphine sulfate.
 28. The method of claim 1 wherein the drug deliverysystem comprises a polymeric matrix.
 29. The method of claim 28 whereinthe drug delivery system is non-disperse.
 30. The method of claim 28wherein the polymeric matrix is selected from the group consisting of apolylactide, a polyglycolide, a polyester, a polyurethane, a polyamide,and combinations thereof.
 31. The method of claim 29 wherein thepolymeric matrix is in a form selected from the group consisting of asphere, a cylinder, and a slab.
 32. The method of claim 28 wherein thedrug delivery system is a dispersion system.
 33. The method of claim 32wherein the polymer matrix is in a form of microspheres.